Date of Award

12-2003

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Microbiology

Advisor

Carol H. Kim

Second Committee Member

Bruce L. Nicholson

Third Committee Member

Robert E. Cashon

Abstract

Type I interferons (IFNs) represent a family of biological molecules whose antiviral, antitumor, and immunomodulatory role is well known. IFNs were first identified in the 1950's and have since been used extensively for the treatment of various cancers, and viral infections. In order to more fully characterize the IFN response, it is often necessary to use animal models. Although the mouse has been used extensively for IFN studies, a lower order vertebrate model is also desirable, as it would provide information about the structure and function of a more ancestral IFN. To this end, herein is described the cloning and characterization of an IFN gene from the zebrafish, Danio rerio, as well as the IFN-inducible gene Mx. Zebrafish IFN (zflFN) has a nucleotide sequence of 558 bases in length, with a deduced amino acid sequence 185 residues in length. Alignment with known IFN sequences reveals a low but significant similarity at the amino acid level, indicating the distant evolutionary relationship of zflFN to mammalian IFNs. To further characterize zflFN, zebrafish liver cells in culture were treated with the synthetic double-stranded RNA molecule polyinosinic:polycytidylic acid (Poly IC), which acts as a viral mimetic and thus an IFN-inducer. Analysis of messenger RNA (mRNA) levels at various times post-induction revealed maximal expression of zflFN mRNA at six and 12 hours postinduction, with a dramatic decrease to basal expression levels by 24 hours. This expression profile fits the pattern of early induction and rapid degradation of mRNA that is a hallmark of higher order vertebrate IFNs, and thus lends hrther support to the role of zflFN as an evolutionary precursor to mammalian IFN. To demonstrate the antiviral activity of zfIFN, zebrafish cells were transiently transfected with an expression construct containing zfIFN DNA and subsequently infected with virus. Cells transfected with zflFN showed a 36% reduction in the number of plaques formed, compared to cells that were not treated with zfIFN. Having determined the validity of zflFN as a true member of the IFN family the next step was to characterize the regulatory effect of zflFN on the zebrafish antiviral gene Mx (zfMx). Zebrafish liver cells produced high levels of zfMx mRNA in response to induction by Poly IC, with peak expression at 24 hours post-induction, indicating upregulation of zfMx by zflFN. To further characterize this regulation, the zfMx promoter region was cloned and inserted upstream of a reporter gene. Addition of zfIFN to cells transfected with the zfMx promoter resulted in high level expression of the reporter gene. Examination of the zfMx promoter revealed the presence of two DNA elements known to bind IFN-inducible transcription factors. Deletion of these elements from the zfMx promoter led to a marked reduction in reporter gene expression, demonstrating the importance of these elements in zflFN-induced upregulation of zfMx. Together, these data definitively prove the existence of IFN in a lower order vertebrate, as well as provide a mechanism for the regulation of zfMx by zflFN. Conservation of this pathway throughout evolution indicates its success in dealing with viral invasion.

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